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Cloning, Expression, and Characterization of Para-Aminobenzoic Acid (PABA) Synthase from Agaricus bisporus 02, a Thermotolerant Mushroom StrainS
Cloning, Expression, and Characterization of Para-Aminobenzoic Acid (PABA) Synthase from Agaricus bisporus 02, a Thermotolerant Mushroom StrainS
Journal of Microbiology and Biotechnology. 2015. Jan, 25(1): 66-73
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : May 22, 2014
  • Accepted : August 11, 2014
  • Published : January 28, 2015
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About the Authors
Li-Xin Deng
State Key Laboratory of Cellular Stress Biology, School of Life Science, Xiamen University, Xiamen, Fujian 361102, P.R. China
Yue-Mao Shen
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, P.R. China
Si-Yang Song
State Key Laboratory of Cellular Stress Biology, School of Life Science, Xiamen University, Xiamen, Fujian 361102, P.R. China
sysong@xmu.edu.cn

Abstract
The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST- Ab ADCS or His- Ab ADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. Ab ADCS has maximum activity at a temperature of approximately 25℃ and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H 2 O 2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.
Keywords
Introduction
Folate, composed of a pteridine ring, para -aminobenzoic acid (PABA), and glutamic acid(s), is an essential cofactor for all living cells and plays critical roles in a diverse range of metabolic pathways, mainly in one-carbon transfer reactions such as amino acid interconversions, and purine and pyrimidine biosynthesis [22] . During the past decade, more and more details were disclosed for de novo synthesis of folate from bacteria and plants; however, little was known about it from mushroom [1 , 3 , 9] . In Escherichia coli , there are three enzymes required for the conversion of chorismate to PABA. PabA is an aminase that supplies ammonia from glutamine hydrolysis; PabB is a member of a family of structurally similar chorismate-utilizing enzymes that catalyze the amination of chorismate, yielding 4-amino-4-deoxychorismate (ADC); PabC is a pyridoxal phosphate-dependent enzyme that catalyzes the elimination of pyruvate from ADC, forming PABA [4] . In most bacteria, pabA and pabB are isolated genes, but pabA and pabB homologs are found as one fused gene in a number of actinomycetes and all of the eukaryotes analyzed so far [6 , 8 , 12 , 23] . PabA and PabB associate with one another to form the aminodeoxychorismate synthase (ADCS, E.C. 6.3.5.8) ( Fig. 2 A, Step 1), whereas PabC acts as the aminodeoxychorismate lyase (ADCL, E.C. 4.1.3.38) ( Fig. 2 A, Step 2).
Agaricus bisporus (Lange) Imbach is the most frequently cultivated species of edible mushrooms [13 , 14] . In contrast with the commercially cultivated common strain A. bisporus 8213, which requires 16~19℃ during the fruiting period, thermotolerant strain A. bisporus 02 can survive at 25℃ and the maximum temperature of 32℃. Suppression subtractive hybridization (SSH) can be used to compare two mRNA populations and obtain cDNA representing genes that are either overexpressed or exclusively expressed in one population as compared with another [7 , 11 , 19] . During the application of SSH to compare the difference between thermotolerant strain 02 and thermosensitive strain 8213, an EST sequence of 1F6 (GH159019) enriched in the strain 02 transcriptosome was cloned, and then the fulllength of the 1F6 gene named pabS (GenBank Accession No. FJ617437) was cloned and reported for enhancing the thermotolerance of mushroom [18] . To analyze the function of the pabS gene, it is necessary to further identify and characterize its encoding protein in vitro .
In this paper, to understand the gene organization of pabS in detail, the corresponding genomic DNA sequence and its 5’ flanking sequence were further cloned. To characterize its encoding protein, pabS cDNA was cloned into the pGEX-4t-1 vector and its recombinant protein ( Ab ADCS) expressed in E. coli , and then GST- Ab ADCS was purified with glutathione Sepharose 4B. In another protocol, pabS cDNA was also cloned into the pET28a vector, and its recombinant protein ( Ab ADCS) expressed in E. coli , and then His- Ab ADCS was purified with Ni Sepharose 6 Fast Flow. Coupling with recombinant E. coli PabC protein ( Ec ADCL), the recombinant Ab ADCSs (GST- Ab ADCS or His- Ab ADCS) were characterized in vitro .
Materials and Methods
- Substrates and Chemicals
Chorismate and para -aminobenzoic acid were purchased from Sigma (Shanghai, China). Protein molecular marker and Taq DNA polymerase were purchased from Fermentas (Xiamen, China). Gel Extraction Kit, Plasmid Mini Kit I, and Cycle-pure Kit were purchased from OMEGA Bio-Tek (Xiamen, China). Ni Sepharose 6 Fast Flow and Glutathione Sepharose 4B were purchased from GE Healthcare (Xiamen). pMD19-T vector and M-MLV RTase cDNA Synthesis Kit were purchased from TaKaRa (Dalian, China). Recombinant A. bisporus Hsp20 (heat-shock protein 20), PPI (peptidyl-prolyl cis-trans isomerase), and BCAT (branched-chain amino acid aminotransferase) were prepared in our laboratory. All chemicals were reagent grade and all solutions were prepared with MilliQ water.
- Strains and Culture Conditions
A. bisporus strains 02 and 8213 were provided by the Mushroom Research and Development Station, Fujian Academy of Agricultural Sciences, China. E. coli DH5α (TaKaRa, Japan) and BL21 (DE3) RIPL (Stratagene, USA) strains were used in this study for protein expression. The pGEX-4T-1 (GE, Sweden) and pET-28a (+) vectors (Novagen) were used for cloning.
- Extraction ofA. bisporus02 Genomic DNA and Total RNA
A. bisporus 02 mycelia were inoculated into PDA liquid medium at 24℃. After 2 weeks, Erlenmeyer flasks were transferred to 24℃ (non heat stress) or 32℃ (heat stress) incubators for 24 h, and then the mycelia were collected with a sterile gauze and washed with sterile water. Genomic DNA was extracted by the modified CTAB method [20] . Total RNA was extracted according to the specifications of the Trizol kit. Then, cDNA of A. bisporus 02 was obtained by reverse transcription of RNAs according to the instruction for the M-MLV RTase cDNA Synthesis Kit.
- Cloning and Sequencing ofA. bisporus02pabSGenomic DNA and cDNA
The pabS genomic DNA was amplified by ExTaq from A. bispous 02 genomic DNA with primers pabSF and pabSR (Table S1) designed based on the pabS cDNA sequence [17] . The method for amplifying pabS cDNA was the same as for pabS genomic DNA, except with A. bispous 02 total cDNA as the template. The PCR conditions were as follows: 95℃ for 2 min; 30 cycles of 94℃ for 30 sec, 57℃ for 35 sec, and 72℃ for 2.5 min; 72℃ for 10 min. The PCR products were purified and cloned into the pMD19-T vector, and transformed into E. coli DH5α. Finally, positive recombinants were sequenced for cloned PCR fragments.
The sequence of pabS cDNA was subjected to BLAST search at the National Centre for Biotechnological Information (NCBI) for similar sequences ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ), and accordingly representative amino acid sequences were downloaded. Alignments were generated by the ClustalW2 server ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ) and visualized by ESPript 2.2 ( http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ).
- Cloning and Sequencing ofA. bisporus02pabS5’ Flanking Sequence
An attempt was made to obtain the pabS 5’ flanking sequence by self-formed adaptor PCR (SEFA PCR) [25] . PCR amplification of the pabS 5’ flanking sequence of the genomic DNA was first performed using three gene-specific primers ( pabS - Sp1 , pabS - Sp2 , and pabS - Sp3 ) (Table S1) located sequentially on the genomic DNA sequence of the pabS gene (KJ609303). The PCR mixture included 15 µl of 2× GC buffer I, 5 µl of 2.5 mM dNTP, 1.5 U of LA-Taq (TaKaRa), and about 1 µg of A. bisporus 02 genomic DNA, and deionized water was added to 30 µl. After SEFA PCR, a second round of nested PCR was run with the single primer pabS - Sp3 (Table S1). To acquire the specific fragment, the third-round PCR was carried out with primers pabS -SP4 and pabS -SP5 (Table S1). The amplified PCR product was recovered and ligated with the pMD19-T vector, transformed into E. coli DH5α, and sequenced.
- Expression ofA. bisporus02pabScDNA and Purification of the Recombinant ADCS
The A. bisporus 02 pabS cDNA ORF was amplified from pabS -cDNA-pMD19T recombinant E. coli strains with primers pabSS and pabSA (Table S1) and constructed into Eco RI and Sal I sites of the pGEX-4t-1 vector; the ligation products were then transformed into E. coli BL21 (DE3) cells. The recombinant plasmid was designated pabS -pGEX-4t-1 and expressed in the same cells in Luria–Bertani (LB) medium containing 100 µg/ml ampicillin. In another protocol, the A. bisporus 02 pabS cDNA ORF was constructed into Eco RI and Sal I sites of vector pET-28a (+), and the recombinant plasmid was designated pabS -pET28a and transformed into E. coli BL21 (DE3) cells. The transformed cells of E. coli were grown at 37℃ in LB medium containing 50 µg/ml kanamycin to an optical density of 0.6 at 600 nm. The expression of the recombinant protein was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and the strain was grown at 16℃ for 12 h. The cells were harvested by centrifugation at 8,000 × g for 30 min, resuspended with lysis buffer (0.1 M Tris/HCl (pH 7.5), 1 mM L-glutamine, 0.3 M NaCl, and 10% (v/v) glycerol), and then sonicated with an Ultrasonic processor (750 W, 30% amplitude, 3 sec on and 3 sec off for 20 min) to release intracellular proteins. The cell-free extract was centrifuged at 18,000 × g for 15 min to remove cell debris. Finally, GST-tagged A. bisporus ADCS protein (abbreviated GST- Ab ADCS) or His-tagged A. bisporus ADCS protein (abbreviated His- Ab ADCS) was purified on glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow columns according to the manufacturer’s instructions. Eluted fractions containing the highest activity were pooled and concentrated by using a 30 kDa Amicon Ultra (Millipore). Proteins were quantified by the Bradford assay using BSA as the standard [5] .
- Expression of thepabCgene ofE. coliand Purification of the Recombinant ADCL
The PabC gene was amplified from the E. coli BL21 (DE3) genome with sense primer pabCS and pabCA (Table S1) designed according to the E. coli PabC sequence (GenBank No. ACT42987) and constructed into the Bam HI and Xho I restriction sites of the pGEX-4t-1 vector. Recombinant vectors were extracted and transformed into BL21 (DE3) cells for protein expression; GST-tagged E. coli ADCL protein (abbreviated Ec ADCL) was induced by IPTG and purified on a glutathione Sepharose 4B Fast Flow column.
- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli [15] to estimate the protein molecular mass and the purity with a stacking gel (4% polyacrylamide) and a separating gel (10% polyacrylamide).
- Enzymatic Activity ofA. bisporusADCS
The enzymatic activity of A. bisporus ADCS was assayed according to published procedures [2 , 21] with some modifications. The standard reaction system (100 µl) contained 50 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , 5 mM L-glutamine, 50 µM chorismate, 10 µg/ml of the recombinant Ab ADCS, and 20 µg/ml desalted Ec ADCL extract (added as indicated) and was incubated at 25℃ for 10~30 min. The PABA peak was monitored by fluorescence (290 nm excitation, 340 nm emission) and quantified on the basis of a standard. One unit of enzyme activity was defined as the amount of Ab ADCS protein releasing 1 µmol of PABA per minute. To determine the aminotransferase specificity, different ammonia donors, such as glutamine, asparagine, and NH 4 + , were used as substrate and the product PABA was analyzed by HPLC.
- Effects of Environmental Factors on Enzyme Activity
The optimal temperature for enzyme activity was determined at a temperature range of 5-50℃ in increments of 5℃ for 30 min under standard assay conditions. The thermal stability assay was performed by incubating 5 µl of 20 mg/ml enzyme at different temperatures for 1 h. Aliquots were removed and assayed under standard conditions.
For the determination of optimum pH of the enzyme, activities were measured over a pH range of 5.0-10.0 in increments of 0.5 pH units under standard assay conditions. The buffers used were 0.2 M disodium hydrogen phosphate-0.1 M citric acid (pH 5.0-6.5), 0.05 M Tris-hydrochloride (pH 7.0-8.5), and 0.2 M glycinesodium hydroxide (pH 9.0-10.5). The pH stability assay was performed by incubating the enzyme at 4℃ in different pH buffers for 3 h. Aliquots were removed and assayed under standard conditions.
The effects of various metal ions were assayed at 25℃ and pH 8.0, where the enzyme solution was pre-incubated with 5 mM of Mg 2+ , Ca 2+ , Zn 2+ , Cu 2+ , Mn 2+ , Co 2+ , K + , Li + , and Na + as chloride salts individually for 30 min. To determine the effect of organic reagents, dimethylsulfoxide, β-mercaptoethanol, ethanol, and isopropanol at a concentration of 1% (v/v); SDS 0.1% (v/v); EDTA (5 mM); dithiothreitol (5 mM); and PMSF (5 mM) were added to the reaction system individually. The production quantity of PABA was determined by HPLC, where the activity was expressed as percent relative activity with respect to maximum activity.
The effect of H 2 O 2 was assayed by incubating 3.0 mg/ml His- Ab ADCS protein with (A) 5 µmol/l; (B) 50 µmol/l; or (C) 500 µmol/l of H 2 O 2 at 4℃ for 15 min, and the standard reaction condition was used for determination of PABA, where the activity was expressed as percent relative activity with respect to enzyme activity without H 2 O 2 treatment. At 50 µmol/l H 2 O 2 as pretreating concentration, His- Ab ADCS protein was then incubated with 0.9 mg/ml Hsp20, 0.2 mg/ml PPI, 0.25 mg/ml BCAT protein, or 250 mmol/l DTT at 4℃ for 20 min and the recovery of enzymatic activity assayed.
- Kinetic Parameters ofA. bisporusADCS
Enzyme kinetic parameters of Ab ADCS were obtained by measuring the rate of PABA production with one substrate at various concentrations and the other substrate at saturating concentration in the standard reaction condition. For the determination of K m of chorismate, chorismate concentrations ranged from 0 to 500 µM whereas glutamine concentration did not vary (5 mM). For the determination K m of glutamine, L-glutamine concentrations ranged from 0 to 5 mM whereas chorismate concentration did not vary (200 µM). The Michaelis–Menten constant (K m ) and maximum velocity (V max ) values were determined from the Lineweaver–Burk plot.
- Nucleotide Sequence Accession Number
The nucleotide and 5’ flanking sequence of the A. bisporus aminodeoxychorismate synthase gene has been deposited in the GenBank database under the accession numbers KJ609303 and KJ817170.
Results
- Cloning ofA. bisporus02pabScDNA, Genomic DNA, and 5’ Flanking Sequence
Cloning genomic DNA, cDNA, and 5’ flanking sequences of the A. bisporus 02 pabS gene are shown in Fig. S1. Comparing the cDNA and genomic DNA sequences of pabS suggests the presence of two introns that obey GT-AT rule (Fig. S2A). The analysis of the whole gene of pabS cDNA revealed an open reading frame (ORF) encoding a hypothetical 733 amino acid protein with a molecular mass of 80.6 kDa. Clustal analysis of the pabS cDNA corresponding amino acid suggests that a “triad”-family of amidotransferases exists in the N-terminal of pabS of A. bisporus 02; a conserved glutamate (consensus sequence SPERF) required for the cleavage of the C4 hydroxyl group of chorismate and the conserved sequence PI(M)KGT involved in the nucleophilic attack of C2 of chorismate exist in the C-terminus of pabS of A. bisporus 02 (Fig. S2B). The motif was recently believed to allow discrimination of PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active-site motif [4] . Thus, pabS cDNA of A. bisporus 02 encodes a protein containing putative aminodeoxychorismate synthase. The close canonical recognition sequence for the transcription factors TFIID (TATA box) was found approximately 75 bp upstream of the transcription start point. Promoter analysis with TFSEARCH ( http://www.cbrc.jp/research/db/TFSEARCH.html ) suggested it has at least one heat shock factor (HSF) (AGAAC box) binding site fully matching that of yeast (Fig. S3) [10] .
- Expression and Purification ofA. bisporusADCS
For recombinant protein expression vector construction, the ORF of pabS was subcloned ( Fig. 1 A). To analyze the function of the pabS gene of A. bisporus 02 encoding protein, we expressed it in E. coli under the T7 promoter. Overexpression of the cloned pabS ORF induced by IPTG resulted in a high expression of soluble Ab ADCS with N-terminal GST tag or His tag. The GST- Ab ADCS and His- Ab ADCS expressed in E. coli BL21 (DE3) were purified on glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow separately; the molecular mass was determined by comparison with a protein marker as approximately 105 kDa and 82 kDa, respectively ( Figs. 1 C and 1 D).
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Cloning of the AbpabS and EcpabC gene ORFs, and purification of AbADCS and EcADCL proteins. (A-B) Cloning of AbpabS and EcpabC gene ORFs: (A) Lane M, DNA marker; 1, 2, A. bisporus pabS gene ORF; (B) M, DNA marker; 1. pabC gene ORF cloned from E. coli DH5α; 2. pabC gene ORF cloned from E. coli BL21. (C-E) Purification of recombinant protein: (C) GST-AbADCS, (D) His-AbADCS, and (E) GST-EcADCL. M, protein marker; 1, supernatant; 2, pellet; 3, flow-through; 4-5, washed protein; 6-7, eluted protein.
- Expression and Purification ofE. coliADCL
For ADCL protein expression vector construction, the ORF of the E. coli PabC gene was cloned ( Fig. 1 B). After overexpression in E. coli BL21 (DE3), an N-terminal GST tag soluble protein (abbreviated Ec ADCL) was purified on glutathione Sepharose 4B Fast Flow. The molecular mass of Ec ADCL was approximately 55 kDa ( Fig. 1 E).
- Enzymatic Activity ofA. bisporusADCS
The sketch map of PABA production catalyzed by Ab ADCS and Ec ADCL is shown in Fig. 2 A. Representative graphs of HPLC analysis for production are shown in Fig. 2 B: the PABA standard is shown in graph 1; when added without Ab ADCS, and whether with or without Ec ADCL, chorismate was not consumed (graph 2 and 4 separately); when added with Ab ADCS but without Ec ADCL, a little amount of intermediate postulated to be ADC was generated (graph 3); when added both with Ab ADCS and Ec ADCL, PABA was produced (graph 5). Therefore, we confirmed the pabS gene-encoding protein to be 4-amino-4-deoxychorismate synthase. Comparing asparagines, glutamine, and NH 4 + , we believe that glutamine was a natural ammonia donor for Ab ADCS ( Table 1 ).
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PABA synthesis procedure and representative graphs of HPLC analysis. (A) Sketch map of PABA production catalyzed by AbADCS and EcADCL. (B) Representative HPLC analysis graphs for catalyzed PABA production. Graph 1, PABA standard; 2-5, reaction system. 2, without both AbADCS and EcADCL; 3, with only AbADCS; 4, with only EcADCL; 5, with both AbADCS and EcADCL.
Substrate specificity ofAbADCS.
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aThe highest activities of the enzyme for amino donors (L-glutamine) were taken as 100%.
- Effects of Environmental Factors on Enzyme Activity
At pH 8.0, the effect of temperature on Ab ADCS activity was determined; the result showed the optimal temperature of Ab ADCS was approximately 25℃ ( Fig. 3 A). Incubating the protein at different temperatures for an hour suggests, at 5-30℃, Ab ADCS was relatively stable; however, above 35℃, the enzyme activity declined sharply ( Fig. 3 B).
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Effects of temperature and pH on AbADCS enzyme activity. (A) Effects of temperature on enzyme activity. (B) Stability of enzymes after incubating at 5-45℃ for 30 min. (C) Effects of pH on enzyme activity. (D) Stability of enzymes after incubating at pH 5-10.5 for 3 h.
When assayed at various pH values at 25℃, the recombinant Ab ADCS with GST tag or His tag activity showed the optimum pH to be approximately 8.0 ( Fig. 3 C). The enzyme was active and stable in the pH range 7.0-9.0 ( Fig. 3 D).
The effect of metal ions is shown in Fig. 4 A, where in the presence of 5 mM Mg 2+ , Ab ADCS activity reached a maximum, whereas in the same concentration of Mn 2+ , and in Co 2+ or Ca 2+ , its activity was lower; on the contrary, in the presence of the same concentration of Cu 2+ or Zn 2+ , its activity was inhibited. In the presence of monovalent cations such as K + , Li + , Na + , and negative control, a small amount of PABA product was yielded, probably owning to residual Mg 2+ in the extract.
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Effects of metal ions, solvents, and H2O2 on AbADCS enzyme activity. (A) Effects of different metal ions on enzyme activity. 1, Mg2+; 2, Ca2+; 3, Cu2+; 4, Co2+; 5, Mn2+; 6, Zn2+; 7, K+; 8, Li+; 9, Na+; and 10, H2O. (B) Effects of different solvents on enzyme activity. 1. None; 2, DMSO; 3, ethanol; 4, isopropanol; 5, mercaptoethanol; 6, SDS; 7, EDTA; 8, DTT; and 9, PMSF. (C) Effects of H2O2 on His-AbADCS enzyme activity. 1, 5 µmol/l H2O2; 2, 50 µmol/l H2O2; 3, 500 µmol/l H2O2; 4, 50 µmol/l H2O2 + 0.9 mg/ml Hsp20; 5, 50 µmol/l H2O2 + 0.2 mg/ml PPI; 6, 50 µmol/l H2O2 + 0.25 mg/ml BCAT; and 7, 50 µmol/l H2O2 + 250 mmol/l DTT.
The effects of organic solvents are shown in Fig. 4 B, where DMSO did not affect the activity of Ab ADCS; ethanol, isopropanol, and PMSF slightly inhibited the activity, whereas SDS and EDTA strongly inhibited the activity. Of note reducing agents mercaptoethanol and DTT could activate Ab ADCS activity remarkably, which suggests that cysteine residues are essential for its catalytic function.
The damage caused by H 2 O 2 and the recovery by other proteins are shown in Fig. 4 C. Results suggest that a high concentration of H 2 O 2 had serious damage on enzymatic activity; after 50 µmol/l of H 2 O 2 for Ab ADCS pretreating, and then using proteins for recovery, Hsp20 had mild capability for the recovery of the enzyme, whereas PPI and BCAT further damaged the protein. However, DTT recovered most of the activity of the enzyme.
- Kinetic Parameters ofA. bisporusADCS
The initial rates were calculated by measuring the production of PABA at 15 min. By plotting the Lineweaver-Burk double-reciprocal graph, we found that the reaction catalyzed by recombinant Ab ADCS and Ec ADCL proteins obeyed Michealis-Menten kinetics [16] . The resulted plot had a slope equal to K m /V max and an intercept equal to 1/V max . For the substrate chorismate, K m was 116.8~117.0 µM and V max was 12.18~12.64 nmol/(min.mg) of Ab ADCS protein ( Fig. 5 A); for the substrate glutamine, K m was 2.39~2.53 mM and V max was 7.56~11.42 nmol/(min.mg) of Ab ADCS protein ( Fig. 5 B). As the His tag had a far smaller molecular size than the GST tag, we postulate the character of recombinant His- Ab ADCS be more approaching to natural Ab ADCS than GST- Ab ADCS. Detailed kinetic parameters of recombinant His- Ab ADCS are listed in Table 2 .
Steady-state kinetic parameters for His-AbADCS.
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aThe rates of the reaction are expressed as nmol of PABA produced. Results are the means ± standard deviation for triplicate determinations.
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Lineweaver-Burk plot of AbADCS for the kinetic analysis of the reaction rates. (A) At a series of concentrations for chorismate and 5 mM glutamine. (B) At a series of concentrations for glutamine and 200 µM chorismate.
Discussion
PABA is a precursor for the synthesis of folic acid. As an enzyme cofactor, folic acid is involved in numerous basic biological reactions, including nucleotide biosynthesis, DNA repair, and DNA methylation [26] . It was supposed the pabS -encoding protein in A. bisporus could scavenge the reactive oxygen species (ROS) during heat stress [18] .
Herein, we cloned its genomic DNA and 5’ flanking sequence, expressed its encoding protein in E. coli , and analyzed its enzymatic character in vitro . The genomic DNA of pabS has only two introns, which is distinctly different from other similar number of bases of heat shock proteins, such as A. bisporus Hsp90 (GenBank Accession No. KJ609304), which has six introns, or Hsp70 (GenBank Accession No. KJ609305), which has seven introns; this is probably due to its housekeeping function requiring a more rapid mechanism for RNA splicing.
Preliminarily, we considered the GST tag could promote the solubility of the expressing protein and constructed the pabS -pGEX-4t-1 vector and expressed GST- Ab ADCS. As we were afraid that the GST tag was too big and would affect the Ab ADCS configuration, we used thrombin to cut the tag off; however, the product had so many bands on SDS-PAGE that we suspected the cutting sites were not specific. In another protocol, we subcloned pabS cDNA into the pET28a vector and purified the soluble Ab ADCS with an N-terminal His tag. As a long enzyme digestion procedure will result in enzyme activity loss, we compared the biochemical characters of Ab ADCS with N-terminal GST tag and N-terminal His tag directly. The result was that they had no obvious difference, which suggested that the N-terminal GST tag or N-terminal His tag had no significant effects on Ab ADCS biochemical character.
In our experiment, we found Ab ADCS to be a thermosensitive and low-efficient enzyme for binding with chorismate. The K m value of recombinant Ab ADCS for chorismate was 116.8~117.0 µM, whereas its congeners from Arabidopsis thaliana and E. coli were 1.3 ± 0.2 µM and 4.2 ± 1.4 µM, respectively [21 , 24] ; however, their K m values were calculated at 37℃, a different temperature. To investigate the effect of heat stress, the enzyme was incubated with H 2 O 2 and recovered with other upregulated proteins. Results showed that H 2 O 2 did obvious damage on the enzyme; Hsp20 only recovered a little activity of the enzyme, whereas PPI and BCAT did not recover any activity.
In conclusion, we postulate that, during the mild heat stress, the enzyme may be damaged by the ROS generated from heat stress; however, upregulated heat shock proteins cannot completely recover the activity of the enzyme. To sustain the housekeeping function of the enzyme, the mRNA of pabS has to be upregulated and the protein overexpressed. During further heat stress, the upregulated Ab ADCS cannot match the harm from heat stress, and organisms cannot survive. To disclose the relationship of expression of Ab ADCS with the survival rate of mushroom after heat stress, further experiments are required.
Acknowledgements
We are grateful to Professor Xian-ming Deng for constructive discussions about the manuscript, to Professor Zhong-hui Zheng, Professor Lian-ru Zhang, Professor Yao-jian Huang, and Vice Professor Qing-yan Xu for valuable suggestions, and also to Senior experimentalist Zhi-yu Hu for technical support with the experiment. We would also like to thank Professor Wen Deng, Vice Professor Yan Shi, and Dr. Zhong-lei Lu for their generous help and encouragement. This work was supported by the National Nature Science Foundation of China (NSFC No. 30571259).
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